Supplementary Material for: Bidirectional Impact of Varying Severity of Acute Kidney Injury on Calcium Oxalate Stone Formation

Introduction: Acute Kidney Injury (AKI) is a prevalent renal disorder. The occurrence of AKI may promote the formation of renal calcium oxalate stones by exerting continuous effects on renal tubular epithelial cells. We aimed to delineate the molecular interplay between AKI and nephrolithiasis.
Methods: A mild (20 min) and severe (30 min) renal ischemia-reperfusion injury model was established in mice. Seven days after injury, calcium oxalate stones were induced using glyoxylate (Gly) to evaluate the impact of AKI on the formation of kidney stones. Transcriptome sequencing was performed on tubular epithelial cells (TECs) to elucidate the relationship between AKI severity and kidney stones. Key transcription factors (TF) regulating differential gene transcription levels were identified using motif analysis, and pioglitazone, ginkgetin, and fludarabine were used for targeted therapy to validate key transcription factors as potential targets for kidney stone treatment.
Results: Severe AKI led to increased deposition of calcium oxalate crystals in renal, impaired kidney function, and upregulation of kidney stone-related gene expression. In contrast, mild AKI was associated with decreased crystal deposition, preserved kidney function, and downregulation of similar gene expression. Transcriptomic analysis revealed that genes associated with inflammation and cell adhesion pathways were significantly upregulated after severe AKI, while genes related to energy metabolism pathways were significantly upregulated after mild AKI. An integrative bioinformatic analysis uncovered a TF regulatory network within TECs, pinpointing that PKNOX1 was involved in the upregulation of inflammation-related genes after severe AKI, and inhibiting PKNOX1 function with Pioglitazone could simultaneously reduce the increase of calcium oxalate crystals after severe AKI in kidney. On the other hand, motif analysis also revealed the protective role of STAT1 in the kidneys after mild AKI, enhancing the function of STAT1 with Ginkgetin could reduce kidney stone formation, while the specific inhibitor of STAT1, Fludarabine, could eliminate the therapeutic effects of mild AKI on kidney stones.
Conclusion: Inadequate repair of tubular epithelial cells after severe AKI increases the risk of kidney stone formation, with the upregulation of inflammation-related genes regulated by PKNOX1 playing a role in this process. Inhibiting PKNOX1 function can reduce kidney stone formation. Conversely, after mild AKI, effective cell repair through upregulation of STAT1 expression can protect TEC function, reduce stone formation, and activating STAT1 function can also achieve the goal of treating kidney stones.

引言：急性肾损伤（Acute Kidney Injury, AKI）是一种常见的肾脏疾病。AKI的发生可通过持续作用于肾小管上皮细胞，促进肾草酸钙结石的形成。本研究旨在阐明急性肾损伤与肾结石病之间的分子互作机制。
方法：本研究在小鼠中构建了轻度（20分钟）和重度（30分钟）肾缺血再灌注损伤模型。造模7天后，采用乙醛酸（Gly）诱导草酸钙结石形成，以评估急性肾损伤对肾结石形成的影响。对肾小管上皮细胞（Tubular Epithelial Cells, TECs）进行转录组测序，以解析急性肾损伤严重程度与肾结石之间的关联。通过基序分析鉴定调控差异基因转录水平的关键转录因子（Transcription Factor, TF），并使用吡格列酮、银杏黄素和氟达拉滨进行靶向治疗，以验证关键转录因子作为肾结石治疗靶点的潜力。
结果：重度急性肾损伤可导致肾脏内草酸钙晶体沉积增加、肾功能受损，以及肾结石相关基因表达上调；与之相反，轻度急性肾损伤则伴随晶体沉积减少、肾功能保留及同类基因表达下调。转录组分析显示，重度急性肾损伤后炎症与细胞黏附通路相关基因显著上调，而轻度急性肾损伤后能量代谢通路相关基因显著上调。整合生物信息学分析揭示了肾小管上皮细胞内的转录因子调控网络，明确PKNOX1参与了重度急性肾损伤后炎症相关基因的上调过程；使用吡格列酮抑制PKNOX1功能，可同时减轻重度急性肾损伤后肾脏内草酸钙晶体的增多。此外，基序分析还揭示了STAT1在轻度急性肾损伤后肾脏中的保护作用：通过银杏黄素增强STAT1功能可减少肾结石形成，而STAT1的特异性抑制剂氟达拉滨则可抵消轻度急性肾损伤对肾结石的防治效果。
结论：重度急性肾损伤后肾小管上皮细胞修复不全，会增加肾结石形成风险，其中PKNOX1调控的炎症相关基因上调在该过程中发挥作用；抑制PKNOX1功能可减少肾结石形成。反之，轻度急性肾损伤后，通过上调STAT1表达实现的有效细胞修复可保护肾小管上皮细胞功能、减少结石形成，而激活STAT1功能同样可达到治疗肾结石的目的。